Phylogenetic Analysis of Newcastle Disease Virus Isolated from Poultry in Live Bird Markets and Wild Waterfowl in Zambia.

Poultry production is essential to the economy and livelihood of many rural Zambian households. However, the industry is threatened by infectious diseases, particularly Newcastle disease virus (NDV) infection. Therefore, this study employed next-generation sequencing to characterise six NDV isolates from poultry in Zambia's live bird markets (LBMs) and wild waterfowl. Four NDV isolates were detected from 410 faecal samples collected from chickens in LBMs in Lusaka and two from 2851 wild birds from Lochinvar National Park. Phylogenetic analysis revealed that the four NDVs from LBM clustered in genotype VII and sub-genotype VII.2 were closely related to viruses previously isolated in Zambia and other Southern African countries, suggesting possible local and regional transboundary circulation of the virus. In contrast, the two isolates from wild birds belonged to class I viruses, genotype 1, and were closely related to isolates from Europe and Asia, suggesting the possible introduction of these viruses from Eurasia, likely through wild bird migration. The fusion gene cleavage site motif for all LBM-associated isolates was 112RRQKR|F117, indicating that the viruses are virulent, while the isolates from wild waterfowl had the typical 112ERQER|L117 avirulent motif. This study demonstrates the circulation of virulent NDV strains in LBMs and has, for the first time, characterised NDV from wild birds in Zambia. The study further provides the first whole genomes of NDV sub-genotype VII.2 and genotype 1 from Zambia and stresses the importance of surveillance and molecular analysis for monitoring the circulation of NDV genotypes and viral evolution.

Detection of Old and New World Relapsing Fever Borreliae in Ornithodoros Ticks Collected from Warthog Burrows in Zambia.

Relapsing fever (RF) is an arthropod-borne disease caused by Borrelia spirochete, which is one of the major public health concerns in endemic regions including Africa. However, information on Borrelia spirochetes is limited in Zambia. Here, we investigate the Borrelia spirochetes harbored by Ornithodoros ticks in Zambian National Parks. We analyzed 182 DNA samples pooled from 886 Ornithodoros ticks. Of these, 43 tested positive, and their sequence revealed that the ticks harbored both Old and New World RF borreliae. This research presents the first evidence of Old-World RF borreliae in Zambia. The New World RF borreliae detected herein differed from the Candidatus Borrelia fainii previously reported in Zambia and were closely related to the pathogenic Borrelia sp. VS4 identified in Tanzania. Additionally, Borrelia theileri was recently reported in Zambia. Hence, at least four different Borrelia species occur in Zambia, and the organisms causing relapsing fever there might be more complex than previously thought. We empirically confirmed that real-time PCR with TaqMan minor groove binder probes accurately and simultaneously detected both Old and New World RF. In this manner, they could facilitate quantitative analyses of both types of RF borreliae. Subsequent investigations should endeavor to isolate the aforementioned Borrelia spp. and perform serosurveys on patients with RF.

Antimicrobial Susceptibility Profiles and Molecular Characterisation of Staphylococcus aureus from Pigs and Workers at Farms and Abattoirs in Zambia.

Pigs have been shown to be a reservoir for recently emerging livestock-associated Staphylococcus aureus (LA-SA), including methicillin resistant strains in many countries worldwide. However, there is sparse information about LA-SA strains circulating in Zambia. This study investigated the prevalence, phenotypic and genotypic characteristics of S. aureus from pigs and workers at farms and abattoirs handling pigs in Lusaka Province of Zambia. A total of 492 nasal pig swabs, 53 hand and 53 nasal human swabs were collected from farms and abattoirs in selected districts. Standard microbiological methods were used to isolate and determine antimicrobial susceptibility patterns of S. aureus. Polymerase Chain Reaction was used to confirm the species identity and detect antimicrobial resistance and virulence genes of isolates, whereas genetic diversity was evaluated using spa typing. Overall prevalence of S. aureus was 33.1%, 37.8% for pigs and 11.8% for humans. The isolates were resistant to several antibiotics with resistance ranging from 18% to 98% but were all susceptible to vancomycin. Typical LA-SA spa types were detected. The presence of plasmid mediated resistance genes such as tetM (12.8%), other resistance determinants and immune evasion cluster genes among the isolates is of great public health concern. Thus, continuous surveillance of S. aureus using a "One health" approach is warranted to monitor S.aureus infections and spread of antimicrobial resistance.

Immunization Coverage and Antibody Retention against Rabies in Domestic Dogs in Lusaka District, Zambia.

Rabies remains endemic in Zambia. Despite conducting canine vaccinations in Lusaka district, the vaccination coverage and actual seropositivity in the dog population in Lusaka district are rarely evaluated. This study estimated the seropositivity-based immunization coverage in the owned dog population in Lusaka district using the expanded program on immunization cluster survey method. The time-series trend of neutralizing antibodies against rabies in vaccinated dogs was also evaluated. Of 366 dogs in 200 dog-owning households in Lusaka district, blood samples were collected successfully from 251 dogs. In the sampled dogs, 42.2% (106/251) had an antibody titer ≥0.5 IU/mL. When the 115 dogs whose blood was not collected were assumed to be seronegative, the minimum immunization coverage in Lusaka district's owned dog population was estimated at 29.0% (95% confidence interval: 22.4-35.5). It was also found that a single vaccination with certified vaccines is capable of inducing protective levels of antibodies. In contrast, higher antibody titers were observed in multiple-vaccinated dogs than in single-vaccinated dogs, coupled with the observation of a decline in antibody titer over time. These results suggest the importance of continuous booster immunization to maintain herd immunity and provide useful information to plan mass vaccination against rabies in Zambia.

Domestic dog demographics and estimates of canine vaccination coverage in a rural area of Zambia for the elimination of rabies.

BACKGROUND: An estimated 75% or more of the human rabies cases in Africa occur in rural settings, which underscores the importance of rabies control in these areas. Understanding dog demographics can help design strategies for rabies control and plan and conduct canine mass vaccination campaigns effectively in African countries. METHODOLOGY/PRINCIPAL FINDINGS: A cross-sectional survey was conducted to investigate domestic dog demographics in Kalambabakali, in the rural Mazabuka District of Zambia. The population of ownerless dogs and the total achievable vaccination coverage among the total dog population was estimated using the capture-recapture-based Bayesian model by conducting a canine mass vaccination campaign. This study revealed that 29% of the domestic dog population was under one year old, and 57.7% of those were under three months old and thus were not eligible for the canine rabies vaccination in Zambia. The population growth was estimated at 15% per annum based on the cross-sectional household survey. The population of ownerless dogs was estimated to be small, with an ownerless-to-owned-dog ratio of 0.01-0.06 in the target zones. The achieved overall vaccination coverage from the first mass vaccination was estimated 19.8-51.6%. This low coverage was principally attributed to the owners' lack of information, unavailability, and dog-handling difficulties. The follow-up mass vaccination campaign achieved an overall coverage of 54.8-76.2%. CONCLUSIONS/SIGNIFICANCE: This paper indicates the potential for controlling canine rabies through mass vaccination in rural Zambia. Rabies education and responsible dog ownership are required to achieve high and sustainable vaccination coverage. Our findings also propose including puppies below three months old in the target population for rabies vaccination and emphasize that securing an annual enforcement of canine mass vaccination that reaches 70% coverage in the dog population is necessary to maintain protective herd immunity.

Diversity of trypanosomes in wildlife of the Kafue ecosystem, Zambia.

The Kafue ecosystem is a vast conservation protected area comprising the Kafue National Park (KNP) and the Game Management Areas (GMA) that act as a buffer around the national park. The KNP has been neglected as a potential foci for rhodesiense sleeping sickness despite the widespread presence of the tsetse vector and abundant wildlife reservoirs. The aim of this study was to generate information on circulating trypanosomes and their eminent threat/risk to public health and livestock production of a steadily growing human and livestock population surrounding the park. We detected various trypanosomes circulating in different mammalian wildlife species in KNP in Zambia by applying a high throughput ITS1-polymerase chain reaction (PCR)/nanopore sequencing method in combination with serum resistant associated-PCR/Sanger sequencing method. The prevalence rates of trypanosomes in hartebeest, sable antelope, buffalo, warthog, impala and lechwe were 6.4%, 37.2%, 13.2%, 11.8%, 2.8% and 11.1%, respectively. A total of six trypanosomes species or subspecies were detected in the wildlife examined, including Trypanosoma brucei brucei, T. godfreyi, T. congolense, T. simiae and T. theileri. Importantly we detected human infective T. b. rhodesiense in buffalo and sable antelope with a prevalence of 9.4% and 12.5%, respectively. In addition, T. b. rhodesiense was found in the only vervet monkey analyzed. The study thus reaffirmed that the Kafue ecosystem is a genuine neglected and re-emerging foci for human African trypanosomiasis. This is the first assessment of the trypanosome diversity circulating in free-ranging wildlife of the KNP.

Seroprevalence of Filovirus Infection of Rousettus aegyptiacus Bats in Zambia.

Bats are suspected to play important roles in the ecology of filoviruses, including ebolaviruses and marburgviruses. A cave-dwelling fruit bat, Rousettus aegyptiacus, has been shown to be a reservoir of marburgviruses. Using an enzyme-linked immunosorbent assay with the viral glycoprotein antigen, we detected immunoglobulin G antibodies specific to multiple filoviruses in 158 of 290 serum samples of R aegyptiacus bats captured in Zambia during the years 2014-2017. In particular, 43.8% of the bats were seropositive to marburgvirus, supporting the notion that this bat species continuously maintains marburgviruses as a reservoir. Of note, distinct peaks of seropositive rates were repeatedly observed at the beginning of rainy seasons, suggesting seasonality of the presence of newly infected individuals in this bat population. These data highlight the need for continued monitoring of filovirus infection in this bat species even in countries where filovirus diseases have not been reported.

Molecular epidemiological investigations of plague in Eastern Province of Zambia.

BACKGROUND: Plague is a flea-borne zoonotic and invasive disease caused by a gram negative coccobacillus bacterium called Yersinia pestis. Plague has caused three devastating pandemics globally namely: the Justinian, Black Death and Oriental plague. The disease in the Eastern Province of Zambia has been reported in Nyimba and Sinda Districts in the past 15 years. The aim of this study was to investigate the molecular epidemiology of plague in the two affected districts. Polymerase Chain Reaction (PCR), targeting Plasminogen activator gene (pla gene) of Y. pestis, was performed on suspected human bubo aspirates (n = 7), rodents (n = 216), shrews (n = 27) and fleas (n = 1494). Of these, one positive sample from each source or host was subjected to sequencing followed by phylogenetic analysis. RESULTS: The plasminogen activator gene (pla gene) of Y. pestis was detected in 42.8% bubo aspirates, 6.9% rodents, 3.7% shrew and 0.8% fleas. The fleas were from pigs (n = 4), goats (n = 5) and rodents (n = 3). The sequencing and phylogenetic analysis suggested that the pla gene of Y. pestis in Nyimba and Sinda was similar and the isolates demonstrated a high degree of evolutionary relationship with Antiqua strains from the Republic of Congo and Kenya. CONCLUSION: It can be concluded that pla gene of Y. pestis was present in various hosts in the two districts and the strains circulating in each district were similar and resembles those in the Republic of Congo and Kenya.

Potential Roles of Pigs, Small Ruminants, Rodents, and Their Flea Vectors in Plague Epidemiology in Sinda District, Eastern Zambia.

A cross-sectional study was conducted in the Eastern part of Zambia that previously reported a plague outbreak. The aim of the study was to evaluate the potential role of pigs, goats, and sheep as sero-surveillance hosts for monitoring plague, and to investigate the flea vectors and potential reservoir hosts to establish the current status of plague endemicity in the district. Serum samples were collected from 96 rodents, 10 shrews, 245 domestic pigs, 232 goats, and 31 sheep, whereas 106 organs were eviscerated from rodents and shrews. As for fleas, 1,064 Echidnophaga larina Jordan & Rothschild, 7 Xenopsylla cheopis (Rothschild), and 382 Echidnophaga gallinacea (Westwood) were collected from these animals in 34 villages. Enzyme-Linked Immunosorbent Assay (ELISA) and Polymerase Chain Reaction (PCR) tests were performed on serum, and organs and fleas to determine IgG antibodies against Fraction 1 antigen and pla gene of Yersinia pestis, respectively. ELISA results showed that 2.83% (95% CI = 0.59-8.05) rodents, 9.0% (95% CI = 5.71-13.28) domestic pigs, 4.7% (95% CI = 2.39-8.33) goats, and 3.2% (95% CI = 0.08-16.70) sheep were positive for IgG antibodies against Fra1 antigen of Y. pestis. On PCR, 8.4% (95% CI = 3.96-15.51) of the rodents were detected with Y. pestis pla gene, whereas all fleas were found negative. The common fleas identified were E. larina from pigs, whereas X. cheopis were the only fleas collected from rodents. The presence of sero-positive animals as well as the occurrence of X. cheopis on local rodents suggests that Y. pestis remains a risk in the district.

Molecular characterization of infectious bursal disease viruses detected in vaccinated commercial broiler flocks in Lusaka, Zambia.

Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive viral disease of young chickens and remains one of the economically most important diseases threatening the poultry industry worldwide. In this study, 16 and 11 nucleotide sequences of the VP2 hypervariable region (VP2-HVR) and part of VP1, respectively, of IBD virus (IBDV) detected in vaccinated broiler chickens in Lusaka in 2012 were determined. Phylogenetic analysis revealed that these Zambian IBDVs separated into three genotypes of very virulent (VV) IBDVs. Although the majority of these viruses belonged to the African VV type (VV1), which consisted of viruses from West Africa, South Africa and Zambia, one virus belonged to the East African VV type (VV2). Interestingly, a Zambian IBDV belonging to the VV3 genotype (composed of viruses from several continents) clustered with attenuated vaccine strains. Although sequence analysis of VP2-HVR showed that all detected Zambian IBDVs had conserved putative virulence marker amino acids (i.e., 222A, 242I, 256I, 294I and 299S), one virus had two unique amino acid substitutions, N280S and E300A. This study demonstrates the diversity of Zambian IBDVs and documents for the first time the possible involvement of attenuated vaccine strains in the epidemiology of IBD in Zambia. Strict biosecurity of poultry farms, monitoring of live vaccine use in the field, surveillance and characterization of IBDV in poultry and development of a vaccine from local or regional IBDV field strains are recommended for improved IBD control in Zambia.

Seroepidemiological Prevalence of Multiple Species of Filoviruses in Fruit Bats (Eidolon helvum) Migrating in Africa.

Fruit bats are suspected to be a natural reservoir of filoviruses, including Ebola and Marburg viruses. Using an enzyme-linked immunosorbent assay based on the viral glycoprotein antigens, we detected filovirus-specific immunoglobulin G antibodies in 71 of 748 serum samples collected from migratory fruit bats (Eidolon helvum) in Zambia during 2006-2013. Although antibodies to African filoviruses (eg, Zaire ebolavirus) were most prevalent, some serum samples showed distinct specificity for Reston ebolavirus, which that has thus far been found only in Asia. Interestingly, the transition of filovirus species causing outbreaks in Central and West Africa during 2005-2014 seemed to be synchronized with the change of the serologically dominant virus species in these bats. These data suggest the introduction of multiple species of filoviruses in the migratory bat population and point to the need for continued surveillance of filovirus infection of wild animals in sub-Saharan Africa, including hitherto nonendemic countries.

Metagenomic analysis of the shrew enteric virome reveals novel viruses related to human stool-associated viruses.

Shrews are small insectivorous mammals that are distributed worldwide. Similar to rodents, shrews live on the ground and are commonly found near human residences. In this study, we investigated the enteric virome of wild shrews in the genus Crocidura using a sequence-independent viral metagenomics approach. A large portion of the shrew enteric virome was composed of insect viruses, whilst novel viruses including cyclovirus, picornavirus and picorna-like virus were also identified. Several cycloviruses, including variants of human cycloviruses detected in cerebrospinal fluid and stools, were detected in wild shrews at a high prevalence rate. The identified picornavirus was distantly related to human parechovirus, inferring the presence of a new genus in this family. The identified picorna-like viruses were characterized as different species of calhevirus 1, which was discovered previously in human stools. Complete or nearly complete genome sequences of these novel viruses were determined in this study and then were subjected to further genetic characterization. Our study provides an initial view of the diversity and distinctiveness of the shrew enteric virome and highlights unique novel viruses related to human stool-associated viruses.

A nairovirus isolated from African bats causes haemorrhagic gastroenteritis and severe hepatic disease in mice.

Bats can carry important zoonotic pathogens. Here we use a combination of next-generation sequencing and classical virus isolation methods to identify novel nairoviruses from bats captured from a cave in Zambia. This nairovirus infection is highly prevalent among giant leaf-nosed bats, Hipposideros gigas (detected in samples from 16 individuals out of 38). Whole-genome analysis of three viral isolates (11SB17, 11SB19 and 11SB23) reveals a typical bunyavirus tri-segmented genome. The strains form a single phylogenetic clade that is divergent from other known nairoviruses, and are hereafter designated as Leopards Hill virus (LPHV). When i.p. injected into mice, the 11SB17 strain causes only slight body weight loss, whereas 11SB23 produces acute and lethal disease closely resembling that observed with Crimean-Congo Haemorrhagic Fever virus in humans. We believe that our LPHV mouse model will be useful for research on the pathogenesis of nairoviral haemorrhagic disease.

Detection and characterization of zoonotic pathogens of free-ranging non-human primates from Zambia.

BACKGROUND: Wildlife may harbor infectious pathogens that are of zoonotic concern acting as a reservoir of diseases transmissible to humans and domestic animals. This is due to human-wildlife conflicts that have become more frequent and severe over recent decades, competition for the available natural habitats and resources leading to increased human encroachment on previously wild and uninhabited areas. METHODS: A total of 88 spleen DNA samples from baboons and vervet monkeys from Zambia were tested for zoonotic pathogens using genus or species-specific PCR. The amplified products were then subjected to sequencing analysis. RESULTS: We detected three different pathogenic agents, including Anaplasma phagocytophilum in 12 samples (13.6%), Rickettsia spp. in 35 samples (39.8%) and Babesia spp. in 2 samples (2.3%). CONCLUSION: The continuously increasing contacts between humans and primate populations raise concerns about transmission of pathogens between these groups. Therefore, increased medical and public awareness and public health surveillance support will be required to detect and control infections caused by these agents at the interface between humans and wildlife.

Determination of the prevalence of African trypanosome species in indigenous dogs of Mambwe district, eastern Zambia, by loop-mediated isothermal amplification.

BACKGROUND: Dogs have been implicated to serve as links for parasite exchange between livestock and humans and remain an important source of emerging and re-emerging diseases including trypanosome infections. Yet, canine African trypanosomosis (CAT), particularly in indigenous dogs (mongrel breed) remains under- reported in literature. This study evaluated the performance of loop-mediated isothermal amplification (LAMP) in detecting trypanosomes in blood from indigenous dogs of tsetse-infested Mambwe district in eastern Zambia. METHODS: A cross sectional survey of CAT was conducted within 5 chiefdoms (Msoro, Kakumbi, Munkanya, Nsefu, Malama) of Mambwe district, eastern Zambia, during October 2012. Blood samples from 237 indigenous hunting dogs were collected and screened by microscopy and LAMP. RESULTS: Of the 237 dogs screened for CAT, 14 tested positive by microscopy (5.9%; 95% CI: 2.9 - 8.9%), all of which also tested positive by LAMP. In addition, LAMP detected 6 additional CAT cases, bringing the total cases detected by LAMP to 20 (8.4%; 95% CI: 4.9 - 12.0%). Irrespective of the detection method used, CAT was only recorded from 3 chiefdoms (Munkanya, Nsefu, Malama) out of the 5. According to LAMP, these infections were caused by Trypanosoma congolense, Trypanosoma brucei brucei and the zoonotic Trypanosoma brucei rhodesiense. Although these CAT cases generally did not manifest clinical illness, an association was observed between infection with Trypanosoma brucei subspecies and occurrence of corneal opacity. CONCLUSIONS: This communication reports for the first time the occurrence of CAT in indigenous Zambian dogs. Our study indicates that LAMP is a potential diagnostic tool for trypanosome detection in animals. LAMP was more sensitive than microscopy and was further capable of distinguishing the closely related T. b. brucei and T. b. rhodesiense. In view of the sporadic cases of re-emerging HAT being reported within the Luangwa valley, detection of the human serum resistant associated (SRA) gene in trypanosomes from mongrels is intriguing and indicative of the risk of contracting HAT by local communities and tourists in Mambwe district. Consequently, there is a need for continuous trypanosome surveillances in animals, humans and tsetse flies using sensitive and specific tests such as LAMP.

Molecular epidemiology of paramyxoviruses in Zambian wild rodents and shrews.

Rodents and shrews are known to harbour various viruses. Paramyxoviruses have been isolated from Asian and Australian rodents, but little is known about them in African rodents. Recently, previously unknown paramyxovirus sequences were found in South African rodents. To date, there have been no reports related to the presence and prevalence of paramyxoviruses in shrews. We found a high prevalence of paramyxoviruses in wild rodents and shrews from Zambia. Semi-nested reverse transcription-PCR assays were used to detect paramyxovirus RNA in 21 % (96/462) of specimens analysed. Phylogenetic analysis revealed that these viruses were novel paramyxoviruses and could be classified as morbillivirus- and henipavirus-related viruses, and previously identified rodent paramyxovirus-related viruses. Our findings suggest the circulation of previously unknown paramyxoviruses in African rodents and shrews, and provide new information regarding the geographical distribution and genetic diversity of paramyxoviruses.

Human parainfluenza virus type 3 in wild nonhuman primates, Zambia.

Human parainfluenza virus type 3 (HPIV3) genome was detected in 4 baboons in Zambia. Antibody for HPIV3 was detected in 13 baboons and 6 vervet monkeys in 2 distinct areas in Zambia. Our findings suggest that wild nonhuman primates are susceptible to HPIV3 infection.

Preliminary investigation of trypanosomosis in exotic dog breeds from Zambia's Luangwa and Zambezi valleys using LAMP.

Abstract. Canine African trypanosomosis (CAT) is rarely reported in the literature. In this preliminary study, we evaluated the performance of loop-mediated isothermal amplification (LAMP) against microscopy to detect CAT in six exotic dog breeds naturally infected with trypanosomes from Zambia's South Luangwa National Park and Chiawa Game Management Area. To our knowledge, this is the first report of CAT in Zambia. The patients exhibited a variety of aspecific clinical signs. The LAMP did not only confirm all six parasitologically positive CAT cases detected passively between April 2010 and January 2012, but was also critical in trypanosome speciation. According to LAMP, the majority of the dogs had monolytic infections with either Trypanosoma congolense or Trypanosoma brucei rhodesiense. The LAMP is thus a potential simple and cost-effective tool for trypanosome diagnosis in endemic regions. The rare report of zoonotic trypanosomes in dogs in Zambia has public health implications and justifies further investigations of CAT.

Identification of a novel polyomavirus from vervet monkeys in Zambia.

To examine polyomavirus (PyV) infection in wildlife, we investigated the presence of PyVs in Zambia with permission from the Zambia Wildlife Authority. We analysed 200 DNA samples from the spleens and kidneys (n = 100 each) of yellow baboons and vervet monkeys (VMs) (n = 50 each). We detected seven PyV genome fragments in 200 DNA samples using a nested broad-spectrum PCR method, and identified five full-length viral genomes using an inverse PCR method. Phylogenetic analysis of virally encoded proteins revealed that four PyVs were closely related to either African green monkey PyV or simian agent 12. Only one virus detected from a VM spleen was found to be related, with relatively low nucleotide sequence identity (74 %), to the chimpanzee PyV, which shares 48 % nucleotide sequence identity with the human Merkel cell PyV identified from Merkel cell carcinoma. The obtained entire genome of this virus was 5157 bp and had large T- and small t-antigens, and VP1 and VP2 ORFs. This virus was tentatively named vervet monkey PyV 1 (VmPyV1) as a novel PyV. Comparison with other PyVs revealed that VmPyV1, like chimpanzee PyV, had a longer VP1 ORF. To examine whether the VmPyV1 genome could produce viral proteins in cultured cells, the whole genome was transfected into HEK293T cells. We detected VP1 protein expression in the transfected HEK293T cells by immunocytochemical and immunoblot analyses. Thus, we identified a novel PyV genome from VM spleen.

The use of Loop-mediated Isothermal Amplification (LAMP) to detect the re-emerging Human African Trypanosomiasis (HAT) in the Luangwa and Zambezi valleys.

BACKGROUND: Loop-mediated isothermal amplification (LAMP) is a novel strategy which amplifies DNA with high sensitivity and rapidity under isothermal conditions. In the present study, the performance of the repetitive insertion mobile element (RIME)-LAMP and human serum resistance-associated gene (SRA)-LAMP assays were evaluated using clinical specimens obtained from four male patients from Luangwa and Zambezi valleys in Zambia and Zimbabwe, respectively. FINDINGS: The cases reported in this preliminary communication were all first diagnosed by microscopy, through passive surveillance, and confirmed by both RIME-LAMP and SRA-LAMP. A good correlation between microscopy and LAMP was observed and contributed to staging and successful treatment of patient. RIME-LAMP and SRA-LAMP complimented each other well in all the cases. CONCLUSIONS: Both RIME-LAMP and SRA-LAMP were able to detect Trypanosoma brucei rhodesiense DNA in patient blood and CSF and hence confirmed HAT in the parasitaemic patients. Our study indicates that the LAMP technique is a potential tool for HAT diagnosis, staging and may be useful for making therapeutic decisions. However, no statistically significant conclusion may be drawn due to the limited sample size used in the present study. It is thus imperative to conduct a detailed study to further evaluate the potential of LAMP as a bedside diagnostic test for HAT.